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<p>Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.</p

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<p>Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.</p

Splenocytes from <i>T. cruzi</i> infected mice treated with Tc mucin produce low levels of IFN-γ.

<p>BALB/c mice were infected <i>i.p.</i> with 2×10⁵ chemically induced metacyclic forms of <i>Trypanosoma cruzi</i> Dm28c clone. The mice received <i>i.p.</i> injections of Tc Muc (20 µg/mouse) or PBS on alternate days starting at day of infection. Non-infected mice were used as control group. Twenty four days after infection, purified splenocytes were stimulated with PMA and ionomycin, as described in the Methods section, and supernatants were harvested at 24 h for determination of (<b>A</b>) IFN-γ and (<b>B</b>) TNFα by ELISA. The <i>y</i>-axis represents the levels of cytokines, detected by a specific ELISA assay, expressed in ng/ml. Asterisks represent statistical significance (p<0.05) as determined by the Student t test. To access the T cell activation profile CD69 expression on both CD4⁺ (<b>D</b>) and CD8⁺ (<b>E</b>) T cells were analysed by FACS analysis; the frequency of IFN-γ producing T cells from splenocytes polyclonally stimulated with PMA/ionomycin, and the percentages of both IFN-γ producing CD4⁺ T cells (<b>F</b>) and CD8⁺ T cells (<b>G</b>)<b>,</b> were determined by intracellular cytokine FACS-staining. #Infected group are statistically different from non-infected control mice (<i>P</i>≤0.05). Asterisks represent statistical differences between Tc mucin treated <i>versus</i> non-treated mice from infected groups as determined by the Student t test (<i>P</i>≤0.05). All experiments were repeated at least 3 times.</p

Tc Muc induced G1 cell cycle arrest of CD4⁺ T cells correlates with upregulation of Cyclin D3 and downregulation of p27^kip1.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 in the presence or absence of native or desialylated <i>T. cruzi</i> mucins (20 µg/ml). After 3 days, cell cycle analysis (A) was evaluated by flow cytometry based on propidium iodide (PI) intercalation into the cellular chromatin (for details see Materials and methods). The histograms represent the fluorescence intensity of PI for the indicated groups. (B) Flow cytometry cell cycle analysis revealed that the population of cells in the S and G2/M phase was remarkably decreased by Tc muc (<i>P</i>≤0.05). For determination of the cell cycle checkpoint regulators, cells were harvested after 3 days and whole-cell lysates were prepared for immunoblotting with specific atibodies against cyclin D3, p27^kip1, and actin antibodies used to assure uniform loading (bottom row). Optical densitometry of the western blots used NIH Image software, where cyclin D3 and p27^kip1 expression was normalized with the actin expression. (C) Expression of cyclin D3 was increased in anti-CD3 activated CD4⁺ T cells as compared to controls (<i>P≤</i>0.05); this increase was not observed when stimulation was done in the presence of Tc Muc (<i>P</i> = 0.0383); *the sialylation abolished the property of Tc Muc to induce downmodulation of cyclin D3 expression (<i>P = </i>0.0067). Expression of p27^kip1 was decreased in anti-CD3 activated CD4⁺ T cells as compared to controls (<i>P≤</i>0.05); this decrease was not observed when stimulation was done in the presence of Tc Muc (<i>P</i>≤0.05); # the sialylation abolished the property of Tc Muc to induce upregulation of p27^kip1 expression (<i>P≤</i>0.05). These data are representative of two independent experiments.</p

Tc Muc inhibits anti-CD3 restimulation of activated CD4⁺ T cells.

<p>Naϊve splenic CD4⁺ T cells were stimulated in 48-well culture plates coated with anti-CD3 (5 µg/mL) in the presence or absence of Tc Muc (20 µg/mL) or the same amount of the bovine mucin as control. After 72 hr of stimulation, activated CD4⁺ T cells were harvested and restimulated for an additional 3 days with plate-bound anti-CD3 in the presence or absence of the TcMuc (20 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. *Differences between Tc mucin treatment <i>versus</i> anti-CD3 stimulated positive control are significant (<i>P≤</i>0.05). The data represented above are representative of one of three experiments with similar results.</p

Inhibition of CD4⁺ T cell proliferation is partially recovered upon neuraminidase-treatment of <i>T. cruzi</i> mucin.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 for 72 hr, in the presence or absence of increasing concentrations of native or desialylated Tc Muc (10 and 20 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. *Differences between native or desialylated Tc Muc treatment <i>versus</i> anti-CD3 stimulated positive controls are significant (<i>P≤</i>0.05). #The inhibition of proliferation by Tc Muc was partially recovered when <i>T. cruzi</i> mucin was desialylated by previous treatment with neuraminidase (<i>P</i> = 0.0023). Results are the means ±SE of triplicate cultures. This experiment was repeated three times, with similar results each time.</p

Tc Muc inhibits CD4⁺ T cell proliferation.

<p>(<b>A</b>) Purified CD4⁺ T cells from naïve spleens were stimulated with pre-coated anti-CD3 for 72 hr, in the presence or absence of increasing concentrations of Tc Muc (10, 20 and 50 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. (<b>B</b>) The inhibition of proliferation by Tc mucin was not observed when control mucin derived from bovine submaxillary glands was used, nor was it reverted by addition of exogenous IL-2 when naïve splenic purified CD4⁺ T cells were stimulated with pre-coated anti-CD3 for 72 hr. Results are the means ±SE of triplicate cultures of three different experiments. *Differences between Tc mucin treatment <i>versus</i> anti-CD3 stimulated positive control are significant (<i>P≤</i>0.05).</p

Tc mucin inhibits cytokine production upon TCR stimulation.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 (5 µg/mL), in the presence or absence of Tc Muc (20 µg/mL). Cytokines IL-2, IL-4, IL-10, IFN-γ, TNF-α and TGF-β were detected by ELISA in the supernatants obtained after 48 h stimulation. All cytokine values in the presence of Tc Muc were significantly lower than controls (<i>P≤</i>0.05). Results are the means ±SD of triplicate cultures of three different experiments.</p

<i>T. cruzi</i> infected mice develop a higher parasitemia and reduced heart inflammatory infiltration when treated with Tc Muc.

<p>BALB/c mice were infected via <i>i.p.</i> with 2×10⁵ chemically induced metacyclic forms of <i>Trypanosoma cruzi</i> Dm28c clone. The mice received <i>i.p.</i> injections of Tc Muc (20 µg/mouse) or PBS on alternate days starting at day of infection. (A) The parasitemia for each mouse group was represented as the mean ± the standard deviation (SD) (<i>n</i> = 5). The parasitemia of mice from the Tc Muc treated group was significantly higther than untreated control mice (<i>P</i>≤0.01). (B and C) Inflammatory infiltration is diminished in the heart by treatment with Tc Muc. Twenty four days after infection cardiac fragments were extracted from <i>Trypanozoma cruzi</i> infected mice (B, 1–2) untreated or (B, 3–4) treated with Tc Muc. Slides were stained with haematoxylin-eosin and cellular nuclei from inflammatory and resident cells counted using Leica QWin program in sections with different magnifications, 40× (B, left panels) and 100× (B, right panels). (C) Inflammatory indexes were determined by counting inflammatory foci (average counts per field). Data were obtained from survivors (2 independent experiments) and shown as mean/standard error of the mean. Asterisk (<i>P</i>≤0.05) means statistical difference between infected mice treated with Tc mucin <i>versus</i> infection control group.</p

Triggering of the Tc Muc ligand Siglect E (CD33) induces suppression of CD4⁺ T cell proliferation <i>in vitro</i>.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 for 72 hr, in the presence or absence of anti-CD33 or isotype control antibody (40 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. *Differences between Tc mucin or anti-CD33 treatment <i>versus</i> anti-CD3 stimulated positive control are significant (<i>P</i>≤0.05).</p